The central tenet to be investigated in this proposal is that nitric oxide (NO) is a cell signaling molecule and mediator of cytotoxicity is not just dependent upon its instantaneous concentration at a given point, but rather it is also dependent upon its concentration gradient. The protocols that are currently in use for NO research do not allow the spatial release of biologically relevant concentration gradients of NO, nor do they allow the direct temporal and spatial detection of NO with fine resolution. An important goal of this proposal is to develop novel fluorescent probes and donors that afford spatiotemporal detection of NO, and to develop new methods for well defined spatiotemporal release of NO. To achieve a greater understanding of the role of NO gradients in mediating cell signaling and cytotoxicity, the research conducted in this COBRE IDeA proposal will vertically integrate five medically relevant research projects that employ unique but complementary experimental systems. These novel NO reagents will be used throughout this proposal and will be specifically used in this project to test the hypothesis that nitric oxide is a sperm chemotactic molecule. Because NO has been implicated in bowel dysfunction in the elderly, Project 2 will employ an aged rat model system to investigate whether alterations in neural and gastrointestinal NO signaling within the enteric nervous system of the bowel investigate whether alterations in neural and gastrointestinal NO signaling within the enteric nervous system of the bowel are involved. Project 3 will elucidate specific cell physiology, pathology, and signaling pathways involving NO production in the upper airway epithelium and its role in exacerbating and/or moderating disease caused by respiratory tract pathogens. Project 4 will investigate the role of NO in retrovirus-induced neurodegeneration using a murine retrovirus model. This investigation will provide insight into the potential role of NO in HV-induced neurological disease. Finally, Project 5 will examine the role of NO in controlling infection by obligate intracellular bacterial human pathogens. The bacterial response to NO stress will also be investigated. Not only will these studies lead to the development of novel NO release and detection reagents with broad scale applicability, they will also provide a greater understanding of the role of NO as a signaling molecule, and as protective or damaging factor in the progression of human disease.